The invention relates to novel monomeric building blocks for labeling peptide nucleic acids and similarly constructed nucleic acid-binding oligomers possessing groups which are coupled to a nitrogen base and/or to the peptide backbone of the peptide nucleic acid. The invention furthermore relates to peptide nucleic acids which contain at least one labelled monomeric building block.
The chemical insertion of labeling groups, e.g. reporter molecules, into nucleic acids is of great importance for a large number of applications. The requirement here is for couplable groups, e.g. amino groups, to be incorporated into the nucleic acid in a specific manner. Specially modified monomeric nucleotide building blocks, which are compatible with the strategy for synthesizing nucleic acids which is subsequently employed, are prepared for this purpose. Thus, trifluoroacetyl-protected or Fmoc-protected aminoalkyloxy-phosphoramidites are, for example, used for 5xe2x80x2-end-labeling in nucleic acid chemistry (cf., e.g., EP-A-224 578, Coull et al., Tetrahedron Lett. 27 (1986), 3991-3994). Phosphoramidites which contain a trifluoroacetyl-protected or Fmoc-protected amino acid group and a DMTr ether have been used for labeling within a nucleic acid molecule, with it being possible for the parent substances to be of non-nucleotide (EP-A-0 313 219, Nelson et al., Nucleic Acids Res. 17 (1989, 7179-7186) or nucleotide (Ruth, DNA 4 (1985), 93, WO 84/03285) nature. In the case of a nucleotide parent substance, particular positions in the nitrogen base or the sugar are suitable for coupling reporter molecules (cf., e.g., Ruth (1991) Oligodeoxynucleotides with reporter groups attached to the base, in: Oligonucleotides and Analogs; A Practical Approach (F. Eckstein, HRSG), Oxford University Press, Oxford, UK, pp. 255-282 and Manoharan et al., Tetrahedron Lett. 36 (1995) 3647-3650).
Corresponding nucleoside triphosphates which are modified at the level of the nitrogen base or the ribose and which are suitable for enzymically synthesizing a nucleic acid are likewise known (EP-A-0063 879, EP-A-0286 898).
The functional groups which are able to couple reporter molecules can be blocked by a suitable protecting group such that the reporter group can only be coupled after the protecting group has been removed from the synthesized nucleic acid by means of so-called post-labeling. However, a reporter molecule can also be directly coupled onto the functional group provided that it is stable under the conditions pertaining during nucleic acid synthesis and protecting group removal. An amino side group which is able to couple reporter molecules can otherwise also be introduced at internucleotide phosphate groups, with the resulting oligonucleotide phosphite triesters or H-phosphonate being oxidized to the corresponding phosphoramidate using a mono-protected diamine (WO 92/08728 and Agrawal et al., Nucleic Acids Res. 18 (1990), 5419).
In the case of the hybridization of nucleic acids which are coupled to reporter molecules within the strand, the melting temperature, Tm, is generally found to be lower after a complementary nucleic acid counterstrand has been hybridized on. In some cases, this lowering of the melting temperature can lead to problems with regard to the specificity and sensitivity of nucleic acid hybridization methods.
As a result of the higher affinity and selectivity, as compared with customary nucleic acids, in their base pairing with a complementary nucleic acid counterstrand, peptide nucleic acids (PNAs) are gaining ever increasing importance for carrying out hybridization reactions (Egholm et al., J. Am. Chem. Soc. 114 (1992), 1895-1897, WO 92/20 702 and WO 92/20 703). In the PNAs, the sugar phosphate backbone of the nucleic acids is replaced with a peptide backbone, e.g. a 2-aminoethylglycine backbone. The nitrogen bases are coupled on at their central nitrogen atom, e.g. by way of a methylenecarbonyl group. The synthesis of a PNA therefore differs appreciably from that of a DNA since different protecting groups and coupling schemes are required. To date, functional groups for further derivatizations, e.g. for inserting reporter molecules, have been introduced at the N terminus of the last PNA building block either directly or after one or more xcfx89-amino acids have been additionally coupled on. It has also been possible to insert functionalizable groups terminally by incorporating lysine residues at the C terminus and at the N terminus.
Since only a limited number of reporter molecules can be inserted into PNA by means of this terminal labeling, there is a great need for alternative methods for inserting labeling groups into PNA.
The present invention achieves this object by providing novel monomeric building blocks for PNA synthesis, which building blocks allow labeling groups to be inserted within the PNA molecule strand, with the labeling group being coupled to a nitrogen base and/or to a peptide backbone.
Surprisingly, it was observed, in this connection, that when PNAs are labeled on their nitrogen bases and on their peptide backbone there is then either an increase in the melting temperature of hybrids with a nucleic acid as compared with hybrids containing unlabeled PNA strands or else the destabilization is at least weaker than in the case of a corresponding DNA-DNA hybrid which contains a labeled DNA strand. This surprising increase in the melting point leads to an increased specificity in hybridization reactions and enables more stringent washing conditions and/or shorter probes to be used in detection methods.
A first embodiment of the present invention relates to monomeric building blocks for synthesizing nucleic acid-binding peptide oligomers which carry a labeling group, or a group which is able to couple with a labeling group, on the nitrogen base. Such monomers are preferably depicted by compounds of the formula (I): 
in which:
B is a natural or unnatural nitrogen base which optionally carries a protecting group,
L is a labeling group which is preferably selected from signal-emitting groups, intercalators and pharmaceutically active groups or a group which is able to couple with a labeling group and which optionally carries a protecting group,
A, C and D are in each case, independently of each other, chemical bonds or organic radicals,
E is a group which is selected from N, R1N+ or CH, where R1 is an organic radical or hydrogen,
Q is a group NR2Y, where R2 is an organic radical or hydrogen, and Y is a protecting group or a carrier,
I is a group which is selected from COX, CSX, SOX or SO2X, where X is OH, SH, OM, SM or a protecting group, and M is a cation, preferably a metal cation or an ammonium cation, and
n is an integer from 1 to 3, preferably 1.
A, C and D are preferably C1-C10-alkylene, alkenylene or alkynylene radicals which can optionally carry heteroatoms such as O, N, P or halogen and/or substituents.
A is particularly preferably a xe2x80x94(CH2)1xe2x80x94COxe2x80x94 radical, where 1 is an integer from 0 to 5. A is most preferably a xe2x80x94CH2COxe2x80x94 radical. C is particularly preferably a xe2x80x94(CH2)kxe2x80x94CHRxe2x80x2xe2x80x94 radical, where k is an integer from 0 to 5 and Rxe2x80x2 is hydrogen or the side chain of a naturally occurring amino acid. C is most preferably a xe2x80x94(CH2)2xe2x80x94 radical. D is particularly preferably a (CH2)mxe2x80x94CHRxe2x80x3xe2x80x94 radical, where m is an integer from 0 to 5 and R is hydrogen or the side chain of a naturally occurring amino acid. D Is most preferably a xe2x80x94CH2xe2x80x94 radical. Alternatively, A and B or A and D can also be bridged with each other, i.e. form a common ring structure.
In another aspect, the present invention relates to monomeric building blocks for synthesizing nucliec acid-binding peptide oligomers which contain at least one signal-emitting group on the peptide backbone. These monomeric building blocks are preferably compounds of the formula (II): 
in which:
B, A, C, D, E, Q and I are defined as in the case of the (I) compounds,
L is a labeling group which is preferably selected from signal-emitting groups, intercalators and pharmaceutically active groups,
and n and m are 0 or 1 to 3, with the proviso that the sum of n+m is not 0. n and m are preferably 0 or 1.
In the compounds of the formula (II), the group L is preferably a substituent of D. Particularly preferably, D is a group xe2x80x94CH(Rxe2x80x2xe2x80x94L), where Rxe2x80x2 is an organic radical as previously defined. Rxe2x80x2 is most preferably the radical of the side chain of a naturally occurring amino acid or of the corresponding enantiomeric compound, e.g. lysine. The asymmetric C atom of the group xe2x80x94CH(Rxe2x80x2xe2x80x94L) preferably has the D configuration.
The nitrogen base of the (I) and (II) compounds is any arbitrary natural or unnatural nitrogen base, with all the nitrogen bases which are able to hybridize with a complementary natural nitrogen base on a DNA or RNA molecule being suitable in principle. The nitrogen base B is preferably selected from thymine, uracil, cytosine, adenine, guanine, hypoxanthine, purine, 7-deazapurine, 2,4-diaminopurine, 2,6-diaminopurine, 7-deazaguanine, pseudouracil, pseudocytosine, pseudoisocytosine, N4,N4-ethanocytosine, N6,N6-ethano-2,6-diaminopurine, 5-(C3-C6)-alkynylcytosine, 5-fluoro-uracil or 2-hydroxy-5-methyl-4-triazolopyrimidine, with the nitrogen base optionally carrying a protecting group.
Since numerous variants of the basic structure of PNA monomers are known in the state of the art, the radicals A, C, D, E, Q and I can assume a large number of known meanings. The reader is particularly referred, with regard to the meaning of these radicals, to the documents WO 92/20 702, WO 92/20 703, DE-A-43 31 012, DE-A-44 08 531, DE-A-44 08 533, DE-A-44 25 311 and EP-A-0 739 898. The relevant passages in these documents relating to the meaning of the abovementioned radicals are hereby incorporated into the present description by reference.
The nitrogen base B preferably carries one or more protecting groups, in particular on exocyclic amino functions.
A protecting group according to the present invention is a chemical group which prevents the functional group to which it is bonded from participating in an undesirable chemical reaction. The protecting group can be removed from this functional group without destroying it.
Examples of suitable protecting groups are base-labile protecting groups such as 9-fluoroenylmethoxycarbonyl (Fmoc), 2,2-[bis(4-nitrophenyl)]ethoxycarbonyl (Bnpeoc), 2-(2,4-dinitrophenyl)ethoxycarbonyl (Dnpeoc), 2-(4-nitrophenyl)ethyloxycarbonyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) and 2-methylsulfonylethyloxycarbonyl (Msc). Of these protecting groups, particular preference is given to Fmoc. Furthermore, acid-labile protecting groups of the urethane type, such as tert-butyloxycarbonyl (Boc) and 4-methoxybenzyloxycarbonyl (Moz), or of the trityl type, such as triphenylmethyl (Trt), (4-methoxyphenyl)diphenylmethyl (Mmt), (4-methylphenyl)diphenylmethyl (Mtt) and di-(4-methoxyphenyl)phenylmethyl (Dmt), are also suitable. Of these protecting groups, particular preference is given to Poc, Trt, Mmt, Mtt and Dmt. Finally, protecting groups of the acyl type, such as benzoyl (Z), isobutyryl, acetyl, phenoxyacetyl, 4-(t-butyl)-benzoyl and 4-(t-butyl)-phenoxyacetyl-4-(methoxy)benzoyl, are also suitable. It is expedient to use those protecting groups which are compatible with the proposed strategy of oligomer synthesis.
In compounds of the formula (I), the group L is coupled to the nitrogen base B. The preferred coupling positions are as follows: when the nitrogen base is a pyrimidine base, (C, T, U or an unnatural derivative thereof), the group L may preferably be bonded to the C-5 position. When the nitrogen base is a cytosine or a derivative thereof, the group L may preferably be bonded to the N-4 position. When the nitrogen base is a purine base (A, G or a derivative thereof), the group L may preferably be bonded to the C-8 position. When the nitrogen base is adenine or a derivative thereof, the group L may preferably be bonded to the N-6 position. When the nitrogen base is a purine base, the group L may preferably be bonded to the N-2 position. When the nitrogen base is a 7-diazapurine, the group L may preferably be bonded to the C-7 position. Particular preference is given to the nitrogen base being a pyrimidine base and to the group L being bonded to the C-5 position.
The group L is a group which is preferably selected from signal-emitting groups, intercalators and pharmaceutically active groups or a group which is able to couple with one of the previously mentioned groups. The group L may carry one or more protecting groups insofar as this is necessary for preventing undesirable reactions under the conditions pertaining during the PNA synthesis. The group L is preferably coupled to the nitrogen base B by way of a bond which is stable under conditions in association with which the intermediate protecting groups which are employed for the given synthesis strategy are eliminated. In particular, L is preferably not an exocyclic amino function which is directly protected by a base-labile protecting group. In the present invention, an exocyclic amino function is understood as being an amino function which is located directly (i.e. not by way of a linker) on the heterocycle.
The group L is preferably a signal-emitting group or a reporter molecule. All the previously known signal-emitting groups for polypeptides and nucleotides, in particular non-radioactive signal-emitting groups, are suitable for this purpose. Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, haptens, e.g. digoxigenin, or biotin and derivatives thereof which are able to bind to streptavidin or avidin, are examples of such groups. Furthermore, the labeling group can also be a photoactivatable crosslinking group, e.g. an azido or an azirine group. Metal chelates which can be detected by electrochemoluminescence are particularly preferred signal-emitting groups, with particular preference being given to ruthenium chelates, e.g. a ruthenium (bispyridyl)32+ chelate. Suitable ruthenium labeling groups are described, for example, in EP-A-0580 979, WO 90/053 01, WO 90/11 511 and WO 92/14 138. These documents are hereby incorporated into the present description by reference.
Furthermore, the group L can also be an intercalator which can intercalate into a PNA-nucleic acid hybrid and in this way enable it to be detected, where appropriate. Examples of suitable intercalators are thiazole orange, ethidium bromide and propidium iodide.
The group L can furthermore be a pharmaceutically active group, e.g. an RNA-cleaving group such as an imidazole-containing residue (cf. WO 93/17 7117, DE-A-44 25 311, WO 96/07 667, or a group which is able to improve the pharmacodynamic or pharmacokinetic properties (WO 94/068 15). These documents are hereby incorporated into the present description by reference.
In the case of the compounds of the formulae (I) and (II), labeling groups can, provided they are compatible with the conditions prevailing in association with the given synthesis strategy, be introduced into the monomeric building block before the nucleic acid-binding oligomers are synthesized. Where appropriate, compatibility can be achieved and/or improved by inserting appropriate groups on functional protecting groups, such as amino or OH groups, in the labeling groups. Examples of suitable groups which are stable during oligomer synthesis are luminescent metal chelates, such as ruthenium (bipyridyl)3, biotin or fluorescein.
On the other hand, the group L can also be a radical which is able to couple to a labeling group. Examples of suitable radicals are reactive groups, such as amino groups or active esters, which are preferably connected to the nitrogen base or the peptide backbone by way of suitable linkers. In this case, L in compounds of the formula (I) then preferably has the structure xe2x80x94Rxe2x80x2xe2x80x94NHY, where Rxe2x80x2 is a C2-C10 alkylene, alkenylene or alkynylene radical which optionally contains heteroatoms, and Y is a protecting group. Rxe2x80x2 is particularly preferably a xe2x80x94CHxe2x95x90CHxe2x80x94CH2xe2x80x94 group.
The novel compounds of the formulae (I) and (II) can be employed as monomeric building blocks in the synthesis of nucleic acid-binding peptide oligomers. Nucleic acid-binding peptide oligomers are compounds which are composed of several monomeric building blocks, with the building blocks being linked at least partially by way of peptide bonds or other acid amide bonds (e.g. CONH, CONR2, CSNH, CSNR2, SONH, SONR2, SO2NH or SO2NR2, where R2 is defined as previously). In addition, these oligomers can bind to nucleic acids by way of base pairing. Such base pairing is usually effected by means of hydrogen bonds between complementary nitrogen bases. In principle, nucleic acid-binding oligomers are able to bind to nucleic acids in two ways. In the first case, a strand of the nucleic acid-binding oligomer binds to a selected region of a single-stranded nucleic acid, with a double strand or duplex being formed. In the second case, two molecules of the nucleic acid-binding oligomer can form a complex with the selected region of a nucleic acid, with a triple helix strand or triplex being formed. Peptide nucleic acids as known from WO 92/20 702 are examples of nucleic acid-binding oligomers. However, the present invention does not only cover PNAs having identical, repeating backbone groups; it also covers nucleic acid-binding oligomers in which the backbone consists of different monomeric subunits, as are described in WO 95/14 706. It furthermore covers compounds according to EP-A-0 627 677, which discloses mixed structures composed of peptide-bound monomers and oligonucleotide subunits. It also covers compounds as disclosed in WO 96/20 212 and EP-A-0 700 928. The abovementioned documents are hereby incorporated into the description by reference.
Nucleic acid-binding oligomers which are particularly preferred are peptide nucleic acids according to formula (III): 
in which
n is an integer of at least 3,
x is an integer from 2 to nxe2x88x921,
each of the groups B1 to Bn is a nitrogen base as previously defined,
each of the groups C1-Cn has the meaning (CR6R7)y, preferably CR6R7, CHR6CHR7 or CR6R7CH2, where R6 is hydrogen and R7 is selected from the group consisting of the side chains of naturally occurring alpha-amino acids, or R6 and R7 are independently selected from the group consisting of hydrogen, C1-C6-alkyl, aryl, aralkyl, heteroaryl, hydroxyl, C1-C6-alkoxy, C1-C6-alkylthio, NR3R4 and SR5, where R3 and R4 are as defined below and R5 is hydrogen, C1-C6-alkyl or C1-C6-alkyl which is substituted by hydroxyl, C1-C6-alkoxy or C1-C6-alkylthio, or R6 and R7 together form an alicyclic system or heterocyclic system, or C1-Cn is CO, CS or CNR3;
each of the radicals D1-Dn has the meaning (CR6R7)2, preferably CR6R7, CHR6CHR7 or CH2CR6CR7, where R6 and R7 are as defined before, and y and z are integers of from 0 to 10, where the sum of y+z is at least 2, preferably more than 2 but not more than 10;
each of the radicals G1-Gnxe2x88x921 has the meaning xe2x80x94NR3COxe2x80x94, xe2x80x94NR3CSxe2x80x94, xe2x80x94NR3xe2x80x94SOxe2x80x94 or xe2x80x94NR3SO2 in any orientation, where R3 is as defined below;
each of the radicals A1-An and E1-En is selected such that:
(a) A1-An is a group of the formula (IIIa) (IIIb), (IIIc) or (IIId), and E1-En is N or R3Nxe2x80x2, or
(b) A1-An is a group of the formula (IIId), and E1-En is CH: 
in which:
X is O, S, Se, NR3, CH2 or C(CH3)2,
Y is a single bond, O, S or NR4,
p and q are in each case an integer of from 0 to 5, where the sum p+q is preferably not larger than 5;
r and s are in each case integers of from 0 to 5, where the sum r+s is preferably not larger than 5;
each of the radicals R8 and R9 is selected independently from the group consisting of hydrogen, hydroxyl, amine, halogen, C1-C4-alkoxy, C1-C4-alkylthio and optionally substituted C1-C4-alkyl, where the substituents are preferably selected from hydroxyl, C1-C4-alkoxy or C1-C4-alkylthio groups;
each of the radicals R3 and R4 is selected independently from the group consisting of hydrogen, C1-C4-alkyl which is optionally substituted by hydroxyl or C1-C4-alkoxy or C1-C4-alkylthio, hydroxyl, C1-C6-alkoxy, C1-C6-alkylthio and amine;
Qxe2x80x2 and Ixe2x80x2 are selected independently from the group consisting of NH2, CONH2, COOH, hydrogen, C1-C6-alkyl, O(C1-C6)-alkyl, an amine which is blocked by a protecting group, labeling groups, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleosidediphosphates, nucleosidetriphosphates, oligonucleotides, including oligoribonucleotides and oligodeoxyribonucleotides, oligonucleosides and soluble and insoluble polymers and also nucleic acid-binding groups, and
x1 and y1 is in each case an integer of from 0 to 10, with the compound being such that a group L as previously defined is present at at least one nitrogen base and/or at a position in the peptide backbone.
Most preferably, the nucleic acid-binding oligomers comprise at least one monomeric subunit of the general formula (IV): 
in which:
B is a nitrogen base as previously defined,
k, l and m are, independently, an integer from 0 to 5,
p is 0 or 1, and
R7 is selected from the group consisting of hydrogen and the side chains of naturally occurring alpha-amino acids.
The synthesis of PNA building blocks according to the invention is described using as an example the nitrogen base thymidine, into which an allylamino group, which is able to couple to other groups, e.g. labeling groups such as Ru(bipyridyl)3, was introduced.
It turned out to be difficult to synthesize the thymine PNA building block which was amino-modified in the C-5 position. Two different synthesis strategies are depicted in FIG. 1 and FIG. 2. As seen in FIG. 1, methyl uracil-1-acetate (2) was prepared from uracil (1) and methyl bromoacetate, with the methyl uracil-1-acetate then being saponified with sodium hydroxide solution to give the corresponding sodium salt (3). An attempt to react 2 with Z-protected allylamine 4 by way of an oxidative coupling using Pd(OAc)2 and t-butyl perbenzoate in accordance with the method of Hirota et al., (Synthesis 1987, 495-496), in which the reaction of uracil derivatives and uridine derivatives is described, was not successful. The most usual method in nucleic acid chemistry, i.e. Cxe2x80x94C linking at the C-5 position by means of the direct mercurization of unprotected 2xe2x80x2-deoxyuridine or 2xe2x80x2-deoxycytidine and subsequent alkylation in the presence of olefins (Bergstrom et al., J. Carbohydrates, Nucleosides, Nucleotides 4 (1977), 257; Bergstrom et al., J. Am. Chem. Soc. 98 (1976) 1587; Cook et al., Nucleic Acids Res. 16 (1988), 4077), was not successful either.
Astoundingly, as can be seen from FIG. 2, it was the use of a modification of the Heck reaction (Heck, J. Am. Chem. Soc. 90 (1968), 5518), of all possible options, which led to the desired Cxe2x80x94C linkage. 5-Iodouracil (5) was first of all alkylated with methyl bromoacetate to give methyl 5-iodouracil-1-acetate (6). The Heck reaction of 6 with Z-protected allylamine in the presence of Pd(OAc)2 and triphenylphosphine in absolute acetonitrile was carried out with the exclusion of air and at a superelevated temperature (bath temperature of 110xc2x0 C.), and yielded the compound (7) together with a small portion of the oxidized byproduct (8). The methyl ester (7) was then hydrolyzed to give the free acid (9), which was reacted with methyl 2-Boc-aminoethylglycine (10) in analogy with the method of Dueholm et al., (J. Org. Chem. 59 (1994), 5767). The desired amino-modified thymine PNA building block (11) was obtained after the ester had been subjected to alkaline hydrolysis.
The Z-protected D-Lys-thymine building block 12, which carries a couplable group on the peptide backbone (FIG. 3), is a known compound which has been used up to now for preparing PNA sequences whose solubility is improved.
The PNA was synthesized on an ABI433A peptide synthesizer in the manner described in T. Koch et al., Automated PNA Synthesis, Int. J. Peptide Protein Res., in press. The couplable components, the Boc/Z PNA standard monomers, the Ado linker, the labeling groups Ru(bpy)3-acid, Ru(bpy)3-Lys and biotin, and also the Boc-Gly-derivatized MBHA resin which were used are depicted in FIG. 4. The monomeric building blocks 11 (FIG. 2) and 12 (FIG. 3) were incorporated for labeling within the PNA strand. The PNA oligomers were purified by means of RP18 HPLC. PNA molecules into which the building blocks 11 or 12 had been incorporated were subsequently labelled with Ru(bpy)3-OSu in DMSO and aqueous 0.1 M NaHCO3.
The novel nucleic acid-binding oligomers can, of course, be synthesized in a variety of ways. Thus, a Boc/Z protecting group strategy is used in the examples of the present application, i.e. the intermediate protecting group for the PNA monomeric building blocks is Boc (elimination with trifluoroacetic acid), and the nitrogen bases are protected with Z(benzyloxycarbonyl; elimination using a trifluoromethanesulfonic acid/trifluoroacetic acid mixture). When this synthesis strategy is used, the amino groups which are present on the monomers which have been modified in accordance with the invention can also be protected with Z.
Another synthesis strategy is described in DE-A-44 08 531. This strategy uses weakly acid-labile intermediate protecting groups in the PNA monomer, in particular monomethoxytrityl. The amino groups in the nitrogen bases are protected with protecting groups which are compatible with weak acids, e.g. acyl protecting groups (benzoyl, isobutyryl, acetyl, etc.). When this strategy is used, additional amino acids which are introduced by means of the novel modification should be protected with trifluoroacetyl or Fmoc.
The synthesis strategy described in DE-A-44 08 533 uses base-labile intermediate amino protecting groups for the PNA synthesis, in particular Fmoc. The nitrogen bases are protected with base-compatible protecting groups, e.g. monomethoxytrityl, Boc, etc. The additional amino group, which is introduced by means of the novel modification and which has to be protected permanently during the synthesis, can be protected with monomethoxytrityl or Boc in this case too.
The invention furthermore relates to a nucleic acid-binding peptide oligomer which contains at least one monomeric building block which contains a labeling group which is coupled to a nitrogen base and/or to the peptide backbone. Preferably, the nucleic acid-binding oligomer contains at least one monomeric building block which is selected from compounds from the formulae (I) and (II). It is furthermore preferred that, when the novel oligomer hybridizes with a complementary nucleic acid, (1) the resulting hybrid, which can be a double strand or a triple strand, should have a higher melting point than a hybrid which contains an oligomer which possesses the same sequence but which lacks a labeling group, or (2) there should be weaker destabilization in a PNA-nucleic acid hybrid than in a nucleic acid-nucleic acid hybrid.
The novel oligomer can contain several identical or different labeling groups and, over and above this, can additionally be coupled to other identical or different labeling groups at the N terminus and the C terminus. Particular preference is given to the novel oligomer being a peptide nucleic acid and having a structure of the general formula (III), as previously defined. The PNA preferably contains at least one monomeric building block of the general formula (IV), as previously defined.
The novel nucleic acid-binding oligomers are employed for hybridizing to nucleic acids. Suitable methods in this context are, on the one hand, those for detecting and/or isolating nucleic acids, e.g. diagnostic detection methods. On the other hand, the novel oligomers can, in particular when L is a pharmaceutically active group, also be employed in therapeutic methods, e.g. as antisense molecules.
The invention also relates to reagents and reagent kits for hybridizing with nucleic acids, which kits contain a novel nucleic acid-binding oligomer in addition to other test components.